Intrarenal handling of 99mTc-DMSA is still controversial, particularly in the existence of tubular reabsorption from the tubular fluid. Experiments were performed with micropuncture technique on the rat kidney in an attempt to elucidate this question. METHODS The concentration profile of 99mTc-DMSA along the nephron was measured in fluid from Bowman's space of surface glomeruli and from the proximal and distal tubules collected by micropuncture. Superficial loops of proximal tubules were micropunctured and microperfused with 99mTc-DMSA or [99mTc] pertechnetate for 10 or 20 min at physiological flow rates; the recovery of activity was measured in the final urine. RESULTS Bowman's space urine contained only 14% of the 99mTc activity of arterial plasma, indicating low filtration of 99mTc-DMSA, likely due to high plasma protein binding. Tubular fluid-to-plasma activity ratios of 0.31 in the proximal tubules and 1.31 in the distal tubules suggest that 99mTc-DMSA is neither secreted nor reabsorbed along the nephron. Ninety-eight percent of the 99mTc-DMSA activity was recovered in the final ipsilateral urine, while only 0.5% was found in the urine of the contralateral kidney. CONCLUSIONS A low fraction of 99mTc-DMSA enters the tubule by glomerular filtration and is not reabsorbed from the tubular fluid. Thus, only peritubular extraction by the tubular cell is responsible for renal uptake of 99mTc-DMSA.